Biomass (cellulosic and lignocellulosic) feedstocks and wastes, such as agricultural residues, wood, forestry wastes, sludge from paper manufacture, and municipal and industrial solid wastes, provide a potentially large renewable feedstock for the production of chemicals, plastics, fuels and feeds. Cellulosic and lignocellulosic feedstocks and wastes, composed of carbohydrate polymers comprising cellulose, hemicellulose and pectins, are generally treated by a variety of chemical, mechanical and enzymatic (enzymatic saccharification) means to release primarily hexose and pentose sugars, which can then be converted by microorganisms to useful products.
Enzymatic saccharification of biomass results in accumulation of cellobiose and glucose in the saccharification vessel producing end-product inhibition of exocellulases, slowing the rate of cellulose hydrolysis. Also produced is xylobiose which produces feedback inhibition of xylanases, slowing the rate of hemicellulose hydrolysis. The slowing of the rate of glucan and xylan hydrolysis may be partially relieved by the addition of higher concentrations of enzyme, however, this solution increases the cost of the process.
Previous methods to address the end-product inhibition during enzymatic saccharification include: use of simultaneous saccharification and fermentation (SSF; Takagi, M., et al., In: Process Bioconversion Symposium, 551-571, 1977) wherein the two steps of saccharification and fermentation were combined.
Combining the two process steps in SSF during biofuel production has a lower capital cost and the presence of the biofuel product lowers the risk of fermentation contamination (Wyman, C. E., et al., Biomass Bioenergy, 3: 301-307, 1992). In addition, in this method the formed glucose is consumed by the fermenting microorganism, relieving the end-product inhibition of both β-glucosidase and cellobiohydrolase enzymes. Formation of cellobiose from cellulose as well as its hydrolysis to glucose is thereby accelerated, increasing the rate of conversion of cellulose to glucose. Another approach to relieving glucose end-product inhibition was described in (WO 2006/101832) wherein the reversible conversion of glucose to fructose and xylose to xylulose partially relieved the end-product inhibition.
Klei et al., (Biotechnol. Bioeng. Symp. (1981) 11 Symp. Biotechnol. Energy Prod. Conserve., 3rd, 593-601) discussed use of immobilized β-glucosidase from Aspergillus phoenicis in hollow fiber ultrafiltration membrane cartridges, used as enzyme reactors, whereby hydrolysis of cellobiose occurred. This system was also used for circulation of cellulases from Trichoderma reesei during saccharification of cellulose. Application of hollow fiber membranes allows continuous processing of cellulose and significantly reduces cellulase requirements.
In U.S. Pat. No. 4,220,721, simultaneous saccharification and fermentation was performed by reusable endoglucanase and cellobiohydrolase enzymes adsorbed to a solid support.
CN101173306 describes combined enzymatic hydrolysis and continuous fermentation for acetone and butanol manufacturing from steam-exploded straw in a hollow fiber membrane reactor. It was indicated that the cellulase enzyme used for enzymatic hydrolysis of the straw could be recycled using the membrane reactor for high efficiency and low cost.
Methods described above have each partially improved the saccharification process and minimally relieved the end-product inhibition during saccharification.
The methods disclosed in the art notwithstanding, more efficient methods are needed to address the problem of end-product inhibition of enzymatic saccharification during simultaneous saccharification and fermentation.